The low-affinity Na+/dicarboxylate cotransporter NaDC1 is found on the apical membrane of the renal proximal tubular epithelium, where it plays an important role in the reabsorption of citric acid cycle intermediates, including citrate and succinate, from the tubular lumen. The transport activity of NaDC1 affects such important physiological functions as citrate homeostasis (and the development of kidney stones), the regulation of blood pressure, maintenance of acid-base balance, and the overall metabolic status of the body. In our last project period we identified a bacterial homolog of NaDC1, called SdcS, which provides a unique tool in our studies of the SLC13 family. SdcS is a Na+dependent dicarboxylate transporter from Staphylococcus aureus, with 35% identity to NaDC1. The long-term objective of our research is to determine the structural basis of substrate and cation binding and translocation in the Na+/dicarboxylate cotransporters, and to better understand the mechanism of ion-coupled transport. The research plan for our next project period is designed to elucidate the structure and function of NaDC1 and SdcS in four Specific Aims. Aim 1 will use cysteine-scanning mutagenesis to identify the substrate and cation permeation pathway in NaDC1, focusing on transmembrane helices (TM) 7 and 8, predicted to form part of the substrate access channel. Aim 2 will use epitope insertions to test our new 13-TM structural model of NaDCt. Aim 3 will use SdcS as a model transporter to characterize amino acids that are accessible from the inside of the cell during the transport cycle. Finally, Aim 4 will use mutagenic selection to identify the residues required to transport citrate in SdcS. The experiments in this project should provide important information on the members of the SLC13 family and on the ion-coupled mechanism of transport. Our results should lay the foundations to develop future treatments of disorders involving NaDC1. Relevance to human health: NaDC1 is an important transporter for metabolic intermediates (including succinate and citrate) that mediate many physiological processes;thus, NaDC1 may be involved in diseases such as kidney stones, high blood pressure, and obesity. The bacterial homolog, SdcS, is closely related to NaDC1 and allows us to perform experiments that are not possible with the mammalian transporters. Understanding how NaDC1 and SdcS work will help us to develop treatments for NaDC1-mediated diseases.